The rapid analysis of genetic material for specific target sequences, e.g. for the presence of single nucleotide polymorphisms, the presence of a certain gene, e.g. a resistance gene, or of mRNA requires easy to use, efficient and reliable new tools. The major problem is the need to detect the DNA or RNA of interest directly in small biological samples such as patient blood or plants. These provide the analyte only in minute amounts. In order to reach the required sensitivity an amplification step is usually required wherein either the nucleic acid analyte is amplified prior to analysis or a detection method is used in which the minute detection signal, directly obtained from the DNA/RNA analyte, is amplified.
Methods for the amplification of the nucleic acid analyte include PCR and other nucleic acid amplification protocols. PCR amplification has the major advantage that, within a pool of different DNA strands obtained from the biological material, only the DNA sequence of interest is amplified. This is the basis for the reliable analysis of single genes in complex biological samples. PCR amplification, however, requires complex process steps which, in some cases, are too inconvenient and expensive. Amplification of the detection signal may be achieved by binding an enzyme such a horse radish peroxidase to the analyte, which converts a given substrate continuously into a colored product.
DNA metallization is another way to amplify the detection signal. A single metallic particle attached to DNA/RNA (the nucleus) catalyzes the deposition of ever more metal, which catalyzes further metal deposition [1-9]. The signal induced by metal deposition grows accordingly in an exponential manner. The metal deposition can be detected either electrically, if the analyte is placed within two electrodes, or optically (e.g. with the eye) because the deposited metal gives rise to a black spot e.g. on paper, in the gel, or in the test tube. In principle, metal deposition is the most sensitive detection method because a small metal cluster (nucleus) is sufficient to start the reaction. In practice, however, the sensitivity of the method is limited by unspecific metal nucleation e.g. through impurities in close spatial vicinity to the analyte for example, on the electrodes, in the gel, or on the paper holding the analyte. In fact unspecific metal deposition is the major reason why silver staining of DNA is not routinely used in oligonucleotide analytics. Silver staining is further complicated by the fact that DNA is unable to build the initiation nucleus itself. It requires prior modification. The method of choice today is the reaction of DNA with glutaraldehyde which covalently attaches to the DNA by sequence-unspecific binding to primary amine groups on nucleobase [7]. This adduct, if treated with a silver salt, reduces the Ag+ of the salt to atomic silver which, while bound to the DNA, functions as the required nucleus. Further treatment of the nucleated DNA with silver salts and reducing agents initiates the exponential metal deposition. Another possibility is to exchange the counterions on the DNA strand by Ag+, which is subsequently reduced to give Ag0 nucleation sites. The major disadvantage is that the glutaraldehyde also reacts with impurities or other chemical species close to the analyte, which again induces unspecific silver deposition.
Metal clusters such as Au, Pd particles or Pt-complexes attached to DNA also function as nucleation sites for further metal deposition up to the construction of conducting wires [1-9]. Here the clusters are attached to reactive groups which form a covalent bond with DNA or to units that just intercalate or bind otherwise to DNA/RNA. All these methods label the entire DNA in a biological sample and therefore do not allow sequence-specific marking and hence sequence-specific analysis of a target DNA such as a single gene in a complex biological sample.
The preparation of labelled DNA domains by a telomerase-mediated incorporation of amine-modified nucleoside triphosphates into a primer-initiated modified telomer repeat is described in [8]. The amine-containing telomers are functionalized with activated gold nanoparticle N-succinimidyl esters to yield gold nanoparticle DNA strands. Enlargement of these nanoparticle sites by further metal deposition along the DNA indeed yields rapid growth of the metal clusters up to the construction of DNA templated molecular nanowires. However, the efficiency of amine-modified triphosphate incorporation into the growing telomer end is low and requires triphosphate doping which results in a distribution of nucleation sites. Thus, the procedure does not allow sequence-specific labelling.
Site-specific labelling of DNA has also recently been attempted via a complex lithographic method [4]. According to this method, a partial protection of DNA molecules is effected by binding of RecA. The unprotected DNA sequences are then treated with glutaraldehyde which marks these sequences for metallization. Site-specific reduction of silver ions by the DNA-bound aldehyde functions results in wire formation along these regions. A sequence-specific labelling of nucleic acid molecules in a complex biological sample is not possible, however.
Although these documents demonstrate the interest in new labelling strategies of DNA, the complicated processes involved in order to achieve marking prevents these systems from being used for any real application. In particular, these methods are unable to selectively label DNA or RNA sequences of interest directly in a crude biological sample.
Thus it was an object of the present invention to provide novel methods and reagents which allow a simple, efficient and specific detection of analytes, particularly of nucleic acids in complex biological samples.